JP3234868B2 - Method for producing D-glucoside 3-dehydrogenase and D-glucoside 3-dehydrogenase or its contents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the action of sucrose, D-glucose, D-glucoside, etc.
The present invention relates to a novel enzyme that specifically dehydrogenates at a position to produce a 3-keto sugar, that is, a novel D-glucoside 3-dehydrogenase, its contents, and methods for producing them.

[0002]

BACKGROUND OF THE INVENTION Agrobacterium tumefaciens (Agrobacterium tumef)
aciens) is known to have the ability to produce D-glucoside 3-dehydrogenase (EC 1.1.9).
9.13: “Enzyme Handbook” p73, Asakura Shoten, 1
982), however, since this Agrobacterium tumefaciens infects higher plants and induces a mutation called crown gall, the production of D-glucoside 3-dehydrogenase requires the elimination of this microorganism to the natural world during the manufacturing process. There is a major drawback in the control of phytopathogenic bacteria, which must be reliably prevented.

[0003]

According to the present invention, a microorganism belonging to the genus Cytophaga also has the ability to produce D-glucoside 3-dehydrogenase, and the D-glucoside 3-produced by Agrobacterium tumefaciens described above. It is based on the finding by the present inventors that they have a productivity different from dehydrogenase. In addition, the enzyme-containing substance extracted from the microorganism belonging to the genus Cytophaga before being separated as an enzyme can also exert the enzyme activity of D-glucoside 3-dehydrogenase without the above-mentioned difficulties.

That is, the enzyme of the present invention is a D-glucoside 3-dehydrogenase having the following properties. (1) Action The hydroxyl group at the 3-position of saccharides such as sucrose, D-glucose and D-glucoside is specifically oxidized by dehydrogenation to generate a 3-keto sugar. (2) Substrate specificity A hydroxyl group at position 3 such as sucrose, D-glucose, D-galactose, maltose, α-methyl-D (+)-glucoside is specifically oxidized by dehydrogenation to generate a 3-keto sugar. In addition, it acts on 1,5-anhydro-D-glucitol which is an anhydrous cyclic sugar alcohol. (3) Optimum pH In a buffer having an ionic strength of 0.05, the optimum pH is 8
Having. (4) Stable pH range When treated at 37 ° C. for 30 minutes in a buffer having an ionic strength of 0.05, it exhibits an activity of 80% or more in the pH range of 6 to 9,
It is stable. (5) Range of optimum temperature for action The optimum temperature for action in 25 mM Tris-HCl buffer is 40 to 6
5 ° C., the optimum temperature is about 55 ° C. (6) Inactivation by temperature When treated at 55 ° C. for 30 minutes in a 10 mM phosphate buffer (pH 6.0) containing 0.2% Triton X-100, almost inactivation occurs. (7) Inhibition, activation and stabilization There is no inhibition of activity by calcium ions. No deactivation by sodium ethylenediaminetetraacetate was observed. In the Tris-HCl buffer, the ion concentration is 50 m
Activity is constant in the range of M to 100 mM. (8) Molecular weight According to SDS-polyacrylamide electrophoresis, about 67,
000 to form a band.

Further, the production method of the present invention belongs to the site fur moth genus, and cultured under aerobic conditions a microorganism producing D- glucoside 3- dehydrogenase having the above properties in a medium, the nature of the culture D-glucoside 3 having
-A method for producing D-glucoside 3-dehydrogenase having the above-mentioned properties or a content thereof, which comprises obtaining a dehydrogenase or a content thereof.

[0006] The enzyme of the present invention specifically oxidizes the hydroxyl group at the 3-position of saccharides such as sucrose, D-glucose and D-glucoside by dehydrogenation to produce 3-ketosugar. Dehydrogenase. Also, an anhydrous cyclic sugar alcohol, 1,5-anhydro-
It has a substrate specificity of acting on D-glucitol. The activity of D-glucoside 3-dehydrogenase produced by Agrobacterium tumefaciens is inhibited by calcium ions, whereas the activity of the enzyme of the present invention is not inhibited by calcium ions.

In use, polyacrylamide,
Photocrosslinkable resin, polyurethane resin, kappa carrageenan, sodium alginate, ion exchange resin, semipermeable membrane,
Those immobilized with a high-molecular enzyme modifier or the like can also be used. As an application, in addition to producing the 3-keto sugar itself by specifically dehydrogenating the 3-position of the corresponding sugar such as sucrose, D-glucose, D-glucoside, etc., various analyzes utilizing the production of the 3-keto sugar For example, measurement of the production amount of triketosugar, measurement of the reduction amount of the corresponding sugar, and the like can be mentioned. Quantification of sugars having no hydroxyl group at the 1-position and / or 2-position of glucoside, as a specific example, can be used as a reagent for diagnosing diabetes.

[0008] The microorganism belonging to the genus Cytophaga used in the production method of the present invention may be selected from among conserved strains, may be isolated from nature, or may be a mutant strain thereof. Furthermore, an artificially created product from these strains, for example, a gene involved in the production of the present enzyme is excised from the cells of these strains, inserted into a vector such as a plasmid, and a host transformed with this vector may be used. May be. Escherichia coli (Es)
There are heterologous hosts such as C. cherichia coli and yeast, and homologous hosts such as microorganisms belonging to the genus Cytophaga. The microorganism belonging to the genus Cytophaga in the stock strains includes Cytophaga allergic ATC.
C35408, Cytoferga actiris (C. aq
uatilis) ATCC 2955, Cytofaga
C. aurantiaca ATCC
12208, Cytoferga fluence (C. di
ffluens) ATCC 23144, C. fermentans ATC.
C19072, Cytoferga Flebensis (C.f.
Levensis) ATCC 27944, C. hutchinsoni A
TCC 33406, C. johnsonae ATCC 29583, C. latercula
ATCC 23177, Cytoferga Ricica (Cl.
ytica) ATCC23174, C. marinoflava ATCC1
9326, Cytoferga Salmonicolor (C. sa
lmoniccolor) ATCC 19041, among which the production ability of Cytoferga marinoflava ATCC 19326 is high.

To obtain a large amount of the enzyme, the above microorganism is cultured. It is generally preferable that the culture temperature is about 20 ° C. to 30 ° C., the pH during the culture is about 5 to 8 and the culture time is about 1 to 9 days, but it is appropriate according to a general microorganism culture method. What is necessary is just to culture | cultivate under aerobic conditions suitably with the culture medium of a suitable composition. For example, a solid medium, a liquid medium, or
A synthetic medium, a semi-synthetic medium, and a natural medium may be appropriately used. As for nutrient sources, carbon sources such as starch, starch hydrolyzate, molasses, sucrose, maltose, fructose, glucose, soybean oil, chicken oil, peptone, malt extract, meat extract, yeast extract, soybean powder, defatted soybean Nitrogen sources such as flour, peanut meal, gluten meal, etc., potassium monophosphate, dipotassium phosphate, disodium phosphate, magnesium sulfate, magnesium chloride, ferrous sulfate, calcium carbonate, sodium chloride, etc. Inorganic salts or the like may be appropriately selected as needed.

In separating an enzyme from a culture, a conventional enzyme separation method can be used as it is. For example, cells cultured by a liquid culture method or a solid culture method are separated into a culture solution and cells by filtration or centrifugation, and then water and an aqueous solvent (sodium chloride, hydrochloric acid, acetic acid, citric acid, etc.)
An extract of the enzyme can be obtained by an appropriate extraction operation using an appropriate solution such as an aqueous solution or various buffers.
When the target enzyme is not present in a solubilized state in the cells, the enzyme can be solubilized using a buffer solution containing a surfactant to obtain an enzyme extract. Here, as the surfactant, sodium dodecyl sulfate (S
DS), sodium cholate, sodium deoxycholate (DOC), cetyltrimethylammonium bromide (C16TAB), dodecylpyridinium chloride,
3-[(3-cholamidopropyl) dimethylammonio]
Ionic surfactants such as -1-propanesulfonic acid (CHAPS), polyoxyethylene isooctyl phenyl ether (Triton X series), polyoxyethylene sorbitol ether (Tween series), and polyoxyethylene nonyl phenyl ether (Nonident P40) ) And the like.
"Neochemical Chemistry Laboratory Course 1 Protein I-Isolation / Purification / Properties-" (published in 1990, edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin) p53-p66. do it.

The enzyme is precipitated in the thus obtained enzyme extract using methanol, ethanol, isopropanol, acetone or the like. Alternatively, salting out is performed using magnesium sulfate or the like for the enzyme solution. Next, the obtained precipitate is dry-pulverized and dialyzed. Alternatively, purification is performed by adsorption and desorption using calcium phosphate, alumina, bentonite, ion exchange resin and the like. here,
Purification methods such as a precipitation method using a white precipitant (nucleic acid, tannic acid, phosphorus / tungsten, etc.), a method of removing impurities with heavy metals, a precipitation method at an isoelectric point, and an electrodialysis method may be used. In addition, a uniform crystal enzyme preparation can be isolated by a crystallization method by adding a salt such as amonium sulfate or polyethylene glycol.

[0012] In various processes leading to these separations, enzyme-containing substances are obtained. For example, a culture solution containing cells, viable cultured cells, air-dried cells dehydrated with acetone or the like, disrupted cells, partially purified products purified to various stages, and the like. The higher the degree of purification, the higher the activity, but on the other hand, the higher the production cost. As described above, the present enzyme-containing material does not have any problems with respect to the control of phytopathogenic bacteria, and thus may be appropriately used depending on the application.

The properties of the enzymes obtained from the microorganisms are described below. Action The hydroxyl group at position 3 of saccharides such as sucrose, D-glucose, and D-glucoside is specifically oxidized by dehydrogenation to generate a 3-keto sugar.

Table 1 shows the relative activities for various sugars with the activity for substrate-specific glucose as 100. It also acts on 1,5-anhydro-D-glucitol by recognizing the 3-position hydroxyl group of 1,5-anhydro-D-glucitol which is an anhydrous cyclic sugar alcohol.

[0015]

[Table 1]

Optimum pH and stable pH range A sodium phosphate buffer and a glycine-NaOH buffer adjusted to an ionic strength of 0.05 using glucose as a substrate were adjusted to a pH range of 6 to 10.5, and the influence of pH was examined. The results are shown in FIG. The optimum pH is 8. FIG. 2 shows the results of examining the residual activity after heating at 37 ° C. for 30 minutes in this buffer. It shows a residual activity of 80% or more in the pH range of 6 to 9. In FIG. 2, the reason why the acid side is not shown above pH 6 is that PMS used as an electron acceptor when measuring the activity is unstable in a buffer solution and a reliable value cannot be obtained. is there.

Methods for measuring activity Hayano, K and Fukui, S .; (1967) JB
C, 242 , 3665. Partially modified the method described in (pH
7.0 was changed to pH 8.0, and the reaction temperature was changed from 20 ° C to 37 ° C.
° C and the electron acceptor was changed from DCIP (2,6-dichlorophenolindophenol) alone to a combination of PMS (phenazine methosulfate) and DCIP) to measure the enzyme activity. That is, 77 mM glucose was used as a substrate, and 0.75 mM PMS was used as an electron acceptor.
And 5 μl of the enzyme-containing solution in 20 μl of 25 mM Tris-HCl buffer (pH 8.0) containing 0.75 mM DCIP.
1 was added, mixed quickly, reacted for 3 minutes, and the amount of reduced DCIP produced was measured by the decrease in absorbance at 600 nm (converted from the absorbance coefficient of DCIP at 600 nm at 600 nm). Under the above conditions, 1 μM per minute
The amount of the enzyme that reduces DCIP is 1 unit.

Figure 3 shows the results of examining the temperature dependence of enzymatic activity with hydrochloric acid buffer solution - [0018] The range of glucose acting optimum temperature 25mM Tris as a substrate. The optimum temperature for operation is 40 to 65 ° C, and the optimum temperature is about 55 ° C.

Inactivation by temperature : Dissolved in 10 mM phosphate buffer (pH 6.0) containing 0.2% Triton X-100, treated at each temperature for 30 minutes, and measured the activity using glucose as a substrate. Shown in In the treatment at 4 ° C. to 37 ° C., the activity of 80% or more is maintained and stable, but it is almost completely deactivated by heating at 55 ° C. for 30 minutes.

Inhibition, activation and stabilization No activity inhibition by calcium ions was observed, and no deactivation by sodium ethylenediaminetetraacetate was observed. FIG. 5 shows the result of examining the effect of ion concentration using a Tris-HCl buffer. The activity becomes constant when the ion concentration is in the range of 50 mM to 100 mM, and the activity decreases when the ion concentration is lower than 50 mM.

Since it was difficult to apply the gel filtration method used for measuring the molecular weight of ordinary proteins because it was solubilized from the cell membrane using a molecular weight surfactant, it was measured by SDS-polyacrylamide gel electrophoresis. However, it was estimated to be about 67,000 daltons. Here, when the denaturing conditions of the enzyme by SDS were changed, a band could be formed at around 20,000 dalton, which was considered to indicate the possibility of taking a subunit structure.

Uniformity Fast system manufactured by Pharmacia (PastSy)
fast gel gradient 8-2
Evaluation was performed by polyacrylamide electrophoresis at 5% (manufactured by Pharmacia). Assuming that the distance from the sample insertion portion to the moving interface was Rf = 1.00, formation of a single band was confirmed by silver staining at an Rf value of about 0.44. Immediately after electrophoresis, the gel was
MPMS, 200 μM nitrotetrazolium salt (NB
T), immersed in a 25 mM sodium phosphate buffer (pH 8.0) containing 250 mM glucose and reacted overnight at 37 ° C. in the light-shielded state. The band portion having enzyme activity showed reduced NBT Formed a precipitate, and the activity was confirmed. The band Rf value for which activity was confirmed completely coincided with the Rf value of the single band by silver staining. Thus, the resulting enzyme is an electrophoretically homogeneous preparation.

Enzyme purification method Microorganisms are cultured under culture conditions specified by the ATCC, centrifuged to collect the cells, the cells are disrupted by a French press, and 10 mM
The mixture is stirred in a phosphate buffer (pH 6), centrifuged to remove broken cells, and the precipitate is solubilized with 2% Triton x-100 to obtain an enzyme-containing solution. After passing the enzyme-containing solution through a DEAE Toyopearl column to adsorb the enzyme, the enzyme is eluted with a 10 mM phosphate buffer (pH 6) containing 0.2% Triton X-100 and 0 to 0.5M NaCl. ,
Next, the eluted enzyme was passed through a Phenyl Toyopearl column to adsorb the enzyme, and then Triton X-100 was used.
In 10 mM phosphate buffer (pH 6) containing 0.05%
The enzyme is eluted using a buffer containing 0.5 to 0 M of aCl. Further, the active fraction was passed through a DEAE5PW column to adsorb the enzyme.
The enzyme is eluted with a 10 mM phosphate buffer (pH 6) containing 0% and 0.5M NaCl.

[0024]

【Example】

<Example 1> Cytoferga Marino Flava ATCC1
The 9326, 1l per polypeptone 10 g, yeast extract _{2g, MgSO 4 · 7H 2 O1g} , NaCl25g, in a medium containing glucose 10 g, cultured until 7l / min aeration conditions, the value of OD ₆₆₀ reached 3.0 ( 25 ℃)
After the cells were collected by centrifugation, 3% NaCl
Was washed with a 10 mM potassium phosphate buffer solution (pH 6.0) containing, and crushed by a French press (1000 kgf) three times. Then, in order to remove unbroken cells, centrifugation was performed at 4,000 × g for 20 minutes at 4 ° C. to obtain a supernatant.
xg, ultracentrifugation for 90 minutes to give D-glucoside 3-
A cell membrane fraction (precipitate) having enzymatic activity as a dehydrogenase was obtained. By the way, about the obtained supernatant,
-No enzyme activity as glucoside 3-dehydrogenase.

<Example 2> 2% Triton X-100 was used for the cell membrane fraction (wet weight 50 mg) obtained in Example 1.
1 ml of a 10 mM potassium phosphate buffer solution (pH 6.0) containing D-glucoside 3-
An enzyme-containing liquid having an enzymatic activity as a dehydrogenase was obtained. Incidentally, the cell membrane after the treatment did not show any enzymatic activity as D-glucoside 3-dehydrogenase.

<Examples 3 and 4> D-glucoside 3 was prepared in the same manner as in Example 1 except that sodium cholate and sodium deoxycholate were used instead of Triton X-100. -An enzyme-containing liquid having an enzymatic activity as a dehydrogenase was obtained.
By the way, regarding the cell membrane after the treatment, D-
No enzymatic activity as glucoside 3-dehydrogenase was observed.

[0027]

EFFECT OF THE INVENTION There are no major difficulties in controlling plant pathogens,
Since it can be easily mass-produced, it can be subjected to various analyzes utilizing the production of triketosugar, such as the production of triketosugar, and the quantification of saccharides, and has a great value as a biochemical reagent.

[Brief description of the drawings]

FIG. 1 shows the working pH range of the enzyme according to the present invention.

FIG. 2 shows a stable pH range of the enzyme according to the present invention.

FIG. 3 is a diagram showing the operating temperature range of the enzyme according to the present invention.

FIG. 4 shows a stable temperature range of the enzyme according to the present invention.

FIG. 5 is a graph showing the influence of the ion concentration of the enzyme according to the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 9/02-9/08 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. D-glucoside 3-dehydrogenase having the following properties (1) Action The hydroxyl group at the 3-position of saccharides such as sucrose, D-glucose and D-glucoside is specifically oxidized by dehydrogenation to form a 3-keto sugar. Generate (2) Substrate specificity A hydroxyl group at position 3 such as sucrose, D-glucose, D-galactose, maltose, α-methyl-D (+)-glucoside is specifically oxidized by dehydrogenation to generate a 3-keto sugar. In addition, it acts on 1,5-anhydro-D-glucitol which is an anhydrous cyclic sugar alcohol. (3) Optimum pH In a buffer having an ionic strength of 0.05, the optimum pH is 8
Having. (4) Stable pH range When treated at 37 ° C. for 30 minutes in a buffer having an ionic strength of 0.05, it exhibits an activity of 80% or more in the pH range of 6 to 9,
It is stable. (5) Range of optimum temperature for action The optimum temperature for action in 25 mM Tris-HCl buffer is 40 to 6
5 ° C., the optimum temperature is about 55 ° C. (6) Inactivation by temperature When treated at 55 ° C. for 30 minutes in a 10 mM phosphate buffer (pH 6.0) containing 0.2% Triton X-100, almost inactivation occurs. (7) Inhibition, activation and stabilization There is no inhibition of activity by calcium ions. No deactivation by sodium ethylenediaminetetraacetate was observed. In the Tris-HCl buffer, the ion concentration is 50 m
Activity is constant in the range of M to 100 mM. (8) Molecular weight According to SDS-polyacrylamide electrophoresis, about 67,
000 to form a band. 2. The method according to claim 1 , which belongs to the genus Cytoferga.
A microorganism which produces D-glucoside 3-dehydrogenase is cultured in a medium under aerobic conditions, and the culture is subjected to the method according to claim 1.
The method for producing D-glucoside 3-dehydrogenase or a content thereof according to claim 1 , wherein the D-glucoside 3-dehydrogenase or the content thereof is obtained. 3. The method according to claim 1 , which belongs to the genus Cytoferga.
Roh microorganisms to produce D- glucoside 3-dehydrogenase, site fur moth-Marinofuraba (C.marin
3. The method according to claim 2, characterized in that it is offlava) ATCC 19326. 4. The method according to claim 1 , which belongs to the genus Cytoferga.
After separation of the cell membrane fraction of microorganisms producing Roh D- glucoside 3- dehydrogenase claim by a surfactant
The method according to claim 2 or 3 , wherein the D-glucoside 3-dehydrogenase according to (1) is solubilized and collected.